We are developing Raman microspectroscopy for characterizing amyloid formation of alpha-syn, which is implicated in Parkinsons disease. This direct spectroscopic method reports on intrinsic molecular vibrations such as protein amide bonds, which arise from coupled vibrational modes of the polypeptide backbone. The position and widths of the amide bands depend on the peptide-bond angles and hydrogen-bonding patterns, and therefore, inform on protein secondary structure as well as local environment. Conformations of alpha-helix, beta-sheet, or random coil exhibit characteristic peak maxima, making quantification of structural compositions possible. To demonstrate the utility of Raman spectroscopy as an effective probe of amyloid structure, we examined the effects of pH and ionic strength as well as four PD-related mutations (A30P, E46K, G51D, and A53T) on alpha-syn fibrils. Raman spectral differences were observed in the amide-I, amide-III, and fingerprint regions, indicating secondary structure and tertiary contacts are influenced by pH and to a lesser extent by NaCl. Faster aggregation times appear to facilitate unique fibril structure as determined by the highly reproducible amide-I band widths, linking aggregation propensity and fibril polymorphism. Importantly, Raman spectroscopy revealed molecular-level perturbations of fibril conformation by the PD-related mutations that are not apparent through TEM or limited proteolysis. The amide-III band was found to be particularly sensitive, with G51D exhibiting the most distinctive features, followed by A53T and E46K. Relating to a cellular environment, our data would suggest that fibril polymorphs can be formed in different cellular compartments and potentially result in distinct phenotypes. In expanding our studies, we have coupled the Raman spectrometer with an inverted microscope, which yields both chemical and spatial information within macroscopic amyloid aggregates. Importantly, for the first time we directly compare intrinsic vibrations in pathological versus functional amyloids, further developing our understanding of structural features that define these two amyloid classes. Specifically, we investigated three pathological and two functional amyloids. For pathological amyloids, we chose to study (1) N-terminal acetylated alpha-syn, which is implicated in Parkinsons disease, (2) amyloid beta (1-40), a peptide important in the development of Alzheimers disease and one of the best characterized amyloids to date, and (3) apolipoprotein C-III, an apolipoprotein linked to cardiovascular disease. While previous studies have shown fibrillar alpha-synuclein and Abeta(1-40) form parallel in-register beta-sheets, there is no reported high-resolution structure of apolipoprotein C-III amyloid, which has an atypical ribbonlike morphology, in contrast to the other straight filaments. We also studied two functional amyloids: (1) the repeat domain (residues 315 to 444) of Pmel17, which is important in melanin biosynthesis and suggested to also form parallel in-register -sheet fibrils that are highly pH-dependent and reversible and (2) the prion-domain (residues 218 to 289) of the fungal protein Het-s, which forms a well-defined and highly uniform beta-solenoid conformation. As spontaneous Raman spectra have never been collected for apolipoprotein C-III or repeat domain of Pmel17, our work expanded the use of vibrational spectroscopy for amyloid characterization.